Immunity to infection by the intracellular parasite Leishmania is mediated by sensitized T cells, however, the antigens which they recognize have not been defined nor has direct recognition of infected macrophages by T cells been clearly demonstrated. We have previously shown that the cell-mediated responses of individuals with healed or healing forms of cutaneous leishmaniasis occurs to as many as 50-70 distinct antigens, however, it is unclear whether all the antigens capable of inducing a T cell response are important in protective immunity. Our hypothesis is that endogenous antigens derived from living amastigotes and presented during the early stages of infection are limited, and that these antigens are most involved in protection. We therefore tried to establish whether infected human monocytes could present antigens to autologous T cells, and we compared the responses of Leishmania specific T cells to antigen-pulsed monocytes and to infected monocytes. Using populations of human monocytes 95-100% infected with L. donovani amastigotes, we demonstrated the ability of infected monocytes to present endogenously derived antigen. T cells from cutaneous patients have been further separated into CD4 negative, CD8 negative or gamma delta negative populations, and their responses compared. CD4- cells from old world cutaneous patients lost their response to both infected and antigen pulsed monocytes. New World cutaneous patients responded better to infected monocytes and demonstrated little reduction in proliferative response after CD4 depletion, and the cytokine responses (gIFN and GMCFS) could be reconstituted by addition of IL-2. Thus CD8 cells seem to be activated in some patients and not others, and their activation may be more dependent on antigens produced by living parasites. The antigens recognized by immune T cells on infected cells are being identified by generating T cell lines and clones reactive with autologoulsly infected cells and using these cells to screen fusion proteins selected from cDNA libraries with Kala-azar serum. It is our hope that this system will serve as a model for studying the cell biological aspects of processing and presentation of antigens derived from intracellular parasites.